New [1,2,4]triazolo[4,3-c]quinazolines as intercalative Topo II inhibitors: Design, synthesis, biological evaluation, and in silico studies

Fifteen quinazoline derivatives were designed and synthesized as DNA intercalators. The cytotoxicity of the designed members was assessed against HCT-116 and HepG2 cancer cell lines. In addition, the topoisomerase II (Topo II) inhibitory effect was assessed. Compound 16 was the most cytotoxic and Topo II inhibitor with low cytotoxicity against Vero cells. Compounds 16, 17, and 18 showed significant DNA binding affinities. Compound 16 showed Topo II catalytic inhibitory effect at a concentration of 10 μM. Further mechanistic investigations revealed the capability of compound 16 to induce apoptosis in HCT-116 cells and arrest the growth at the S and G2/M phases. Also, compound 16 showed a significant increase in the level of BAX (2.18-fold) and a marked decrease in the level of Bcl-2 (1.9-fold) compared to the control cells. In silico studies revealed the ability of the synthesized members to bind to the DNA-Topo II complex.


Introduction
The discovery of new anticancer drugs that target DNA and topoisomerase II (Topo II) continues to pique medicinal chemists' attention [1]. DNA intercalators are a significant class of DNA damaging agents [2]. The process of DNA intercalation causes substantial changes in DNA structure, such as elongation and stiffness, as well as changes in the helix twist angle [3]. Because many medications that block Topo II can also intercalate DNA, this class of bioactive anticancer treatments is referred to as intercalative Topo II inhibitors. Many studies have verified the apoptotic impact of intercalative Topo II inhibitors [4]. Typical DNA intercalators are compounds that have the ability to be inserted between nucleotides of DNA to form a DNA-intercalator complex which can be stabilized by different hydrophobic stacking [5]. For high affinity, these compounds should have three pharmacophoric features which have an essential role in binding. i) The first feature is a planar polyaromatic system (chromophore) [6]. The second one is a cationic center to form ionic interaction with the phosphate moiety of the DNA. The third one is the groove binding side chain, which increases the binding affinity of the intercalator through the occupation of the DNA minor groove [7][8][9] (Fig 1).
There are two types of DNA intercalators. I) Classical intercalators, such as doxorubicin I and amsacrine II, that their entire aromatic system is inserted between the base pairs in the DNA helix [10]. These molecules have one groove binding side chain. II) Threading intercalators, such as mitoxantrone III, have a large, auxiliary, not intercalating groove binding side chain on both sides of the molecule. Classically, the flat aromatic system intercalates between base pairs, while bulky substituents are located in a minor and major DNA groove. One of these substituents must "go through" between base pairs causing a larger deformation of the helix structure and partial breakage of the double-stranded DNA (dsDNA) structure [11]. This process has very slow kinetics, and compounds of this type show higher affinity and lower rates of association and dissociation than other intercalators [12].
Topoisomerase II is a crucial cellular enzyme that modifies DNA topology. This enzyme is involved in several replicative processes such as chromosome replication, recombination, transcription, and segregation. It works by breaking the dsDNA and then resealing the breaks that have been produced [13]. Doxorubicin I [14], mitoxantrone II [15], and amsacrine III [16] are well-known examples of intercalative Topo II inhibitors (Fig 1).
For many years, our team synthesized several intercalative Topo II inhibitors which showed promising anticancer activities as compound IV which was evaluated as a classical DNA intercalator and Topo II inhibitor [18]. In addition, a quinoxaline-sulfonamide hybrid (compound V) showed good cytotoxic and Topo II inhibitory activities [17]. Recently, a new generation DNA intercalators and Topo II inhibitors were discovered by our team such as compounds VI [19], VIIa, b [20], VIII [21], and IX [22].
In the current work, compounds (III-IX) were selected to be lead structures in the synthesis of new derivatives. The rationale of our molecular design depended on the lead modification of such compounds to get new scaffolds of threading DNA-intercalators. The [1, 2,4] triazolo [4,3-c]quinazoline moiety was used as a planar aromatic system. Different aliphatic amines were used as the first groove binding side chain. The second groove binding side chain was 4-methylphenyl, 4-chlorophenyl, or 1,1,1-trifluoromethane moieties (Fig 2). The choice of the different derivatives was based on some chemical and biological considerations. The aryl groups might increase the planarity of the molecule for DNA intercalation and increase aromatic-aromatic stacking interaction with the binding site of the receptor. The chloro atom may exert halogen-halogen contacts or halogen bonding interaction with the receptor. A nonpolar methyl group may enhance hydrophobic interactions with the receptor. The trifluoromethyl group was reported to have positive effect on the activity of bioactive molecules. It can enhance several pharmacokinetic and physicochemical properties such as improved metabolic stability and enhanced membrane permeation. Increased binding affinity of fluorinated drug candidates to target protein has also been documented in a number of cases [23]. In this work. trifluoromethyl moiety was added to increase the lipophilicity of the synthesized compounds to facilitate the diffusion of these compounds across the cytoplasmic and nuclear membranes and interact with the DNA.

PLOS ONE
New [1,2,4]triazolo [4,3-c]quinazolines as intercalative Topo II inhibitors 1 H NMR analysis of compound 12a, as a representative example, exhibited the appearance of a characteristic singlet signal for CH 3 at δ 2.41 ppm. Also, it showed two signals for the CH 2 groups at δ 3.85 and 3.79 ppm. Besides, the NH group was detected at δ 4.85 ppm. 13 [4,3-c]quinazoline with trifluoromethyl moiety is beneficial for cytotoxic activity. This may be attributed to the ability of the trifluoromethyl moiety to form extra hydrogen and hydrophobic bonds and with the target receptor and consequently increase the binding affinity and activity. In addition, the trifluoromethyl moiety increases the lipophilic characters of the synthesized compounds which may facilitate the diffusion of these compounds across the cytoplasmic and nuclear membranes and interact with the DNA.
Compound 16 was the most active member showing IC 50 values of 6.29 and 2.44 μM against HepG2 and HCT-116, respectively. The five most active derivatives were arranged in cytotoxic activity in the descending order of 16 >17 >18 > 20 >19. This indicates that the substitution of [1,2,4]triazolo [4,3-c]quinazoline with different amines affects the biological activity in the descending order of propylamine > ethanolamine > dimethylamine > morpholine > cyclohexylamine. This indicates that the increase bulky structure of amines produced negative effect in the cytotoxic activity. This may be attributed to the high a chance of the open chain aliphatic groups to be oriented into the minor groove of the DNA and hence increase the binding affinity and cytotoxic activity. Additionally, compounds 11a, 12b, and 14a showed significant activities against only HCT-116 cell lines with IC 50 values 8.42, 7.03, and 7.66 μM, respectively. Compounds 12a and 13a showed moderate cytotoxic activities against the two cell lines, while compounds 11a, 12b, and 14a showed moderate activities against only HepG2 cell lines. Also, compounds 13b, 14b, and 15a showed moderate activities against only HCT-116. Finally, compounds 11b and 15b showed weak activities against the two test cell lines.
In addition, it was found that the different amines affect the biological activity in the descending order of propylamine > ethanolamine > dimethylamine > morpholine > cyclohexylamine when we compared the cytotoxic activity of compounds 16, 17 [30][31][32]. To detect the proposed mode of action, the Topo IImediated DNA cleavage assay was performed for the most active member 16 against Topo II α and supercoiled pBR322 DNA.
As shown in Fig 3, compound 16 induced relaxation for the DNA at a concentration of 5 μM. In contrast, it blocked the formation of relaxed DNA at 10 μM. In all concentrations, the linear DNA was not formed. This revealed that compound 16 acts as a Topo II catalytic inhibitor at a high concentration (10 μM) and may serve as Topo II poison at low concentration (5 μM). These results are consistence with the previously reported findings by Atwal et al [33]. It was reported that anthracyclines (doxorubicin and epirubicin) and another intercalating Topo II poison (mitoxantrone) can stabilize Topo II-DNA covalent complexes at higher concentrations. On the other hand, they suppress the formation of Topo II-DNA covalent complexes, thus behaving as Topo II poisons at low concentration and inhibitors at high concentration [33].
2.2.6. Cell cycle analysis. The balance between cell proliferation and death must be maintained to maintain tissue homeostasis [34][35][36][37]. To get an additional relationship between the cell cycle and apoptosis, the effect of compound 16, the most active candidate, on cell cycle distribution and apoptosis induction in HCT-116 cells was studied to gain a better understanding of how it inhibits cancer cell proliferation. Therefore, the HCT-116 cell line was treated with compound 16 at a concentration of 2.44 μM (the IC 50 value of compound 16 against the HCT-116 cell) for 48 h then, the cells were harvested, stained with propidium iodide, and analyzed for cell distribution during the various phases of the cell cycle using Flowing software. The flow cytometry data revealed that the percentage of HCT-116 cells decreased at the G1 phase and increased at the sub-G1, S, and G2/M phases. In detail, for the G1 phase, it decreased from 53.75% to 19.05%. On the other hand. In the S phase, it increased from 20.08% to 42.76%. For the G2/M phase, it increased from 25.10% to 36.89%. These results indicate that compound 16 could arrest the cell growth at S and G2/M phases ( Table 2 and Fig 4A). These findings were corroborated by published studies showing that Topo II inhibitors can stop cell development in the G2/M [38]. Since catalytic topo II inhibitors prevent DNA double strand breaks, they also cause cell cycle delay at the G2/M phases by interfering chromosome condensation and segregation during mitosis [39]. In addition, during S phase, topo II is required to resolve topologically linked supercoiled DNA strands while DNA replication forks proceed. Inhibition of enzyme function at this time by a catalytic inhibitor of topoisomerase II has been reported to delay cell cycle progression during the S phase and cause cells to arrest at the G2 phase, delaying entry into mitosis [40].  Table 3 and Fig 4B).

Effects on apoptotic markers (BAX and Bcl-2)
. B-cell lymphoma protein 2 (Bcl-2) is a member of the B-cell lymphoma protein family that plays a key role in tumor formation and suppression of the intrinsic apoptotic pathway [41]. The anti-apoptotic Bcl-2 protein suppresses apoptosis, whereas BAX promotes it (proapoptotic). As a result, the balance between these two contradictory proteins controls cell fate [42].
In this investigation, HCT-116 cells were treated with 2.44 μM (IC 50 value of compound 16) for 48 hours to see its effect on Bcl-2 and BAX levels. In comparison to the control, compound 16 elevated the level of the proapoptotic protein (BAX) by 2.18 times. Furthermore, compound 16 significantly reduced the anti-apoptotic protein Bcl-2 levels by 1.9-fold when compared to the control. Furthermore, compound 16 enhanced the BAX/Bcl-2 ratio by 4.38-fold in comparison to control cells. These findings demonstrated that the BAX/Bcl-2 ratio may promote the apoptotic process ( Table 4 and Fig 5).
2.2.9. In vitro cytotoxicity against normal cell line. The most active candidate 16 was tested against normal cell lines (Vero cells) using. Although Vero cell are not human cell, it was reported that Vero cells can be used as normal cells to validate the toxicity of the synthesized compounds [43]. In this work, compound 16 showed an IC 50 value of 63.36 μM against the tested normal cells. These results indicated that the tested compound possesses high selectivity against tumor cells. comparing its activity against normal Vero cells, its activity was 10-fold and 26-fold against HepG2 and HCT-116 cells, respectively. The prototype VEGFR-2  [44]. This indicates that the toxicity of compound 16 is less than that of sorafenib by about 2.5-fold.    [45]. For amsacrine, its planar aromatic (acridine) moiety was inserted between the nucleotide of the DNA forming nine pi-pi interactions with Thy9, Cyt8, Ade12, and Gua13. The groove binding side chain (N-(4-amino-3-methoxyphenyl)methanesulfonamide) was oriented into the minor groove of DNA forming two hydrogen bonds with Glu522 and Gua13. Also, it formed two hydrophobic interactions with Arg503 and Gua13 (Fig 6).
The results of the docking study revealed that the docked compounds have a similar binding mode to the reference ligand amsacrine. The binding energies of the docked compounds were summarized in Table 5.
All the docked molecules were inserted between the adjacent nucleotides at the active site. These compounds were stabilized in the active site through different interactions. The planar system was involved in hydrophobic stacking with the base pairs of different nucleotides (Thy9, Cyt8, Gua13, and Ade12). The hydrophilic moieties formed several hydrogen bonds with aforementioned nucleotides. Other stabilizing interactions was achieved by the aid of the side chains (groove binding side chains). These moieties were oriented into the minor groove of the DNA forming hydrogen and hydrophobic bonds. The binding modes of the docked molecules were consistence with the reported findings [18-20].
Regarding compound 11a, The planar aromatic system ([1,2,4]triazolo[4,3-c]quinazoline) was inserted between the adjacent nucleotides of DNA forming ten hydrophobic stacking with, Thy9, Cyt8, Gua13, and Ade12. The terminal toluene moiety bound the minor groove of DNA forming two hydrophobic interactions with Ala521and Pro455. Moreover, the propane moiety was oriented into the minor groove with the formation of one hydrophobic interaction with Gua13 (Fig 7).
Compounds 16, 17, and 19 exhibited similar fitting patterns against the active site. The planar aromatic systems ([1,2,4]triazolo[4,3-c]quinazoline) formed ten, eleven, and seven pi-pi interactions, respectively. The side chains of these compounds were oriented towards the minor groove of DNA. The trifluoromethyl moiety of compound 16 formed four hydrogen bonds with Ade12, Cyt8, and Gln778. The ethanolamine moiety of compound 17 formed one hydrogen bond with Glu522. The trifluoromethyl moiety of compound 19 formed two hydrogen bonds with Cyt8 and Gua13 (Figs 9-11).
Most compounds showed in silico low adverse effects and toxicity against the tested models. In general, all compounds were predicted to be non-carcinogenic against the FDA rodent carcinogenicity mouse model.
For carcinogenic potency TD 50 rat model, compounds 11a, b, 12a, b, 16, 17, 18, and 20 showed TD 50 values ranging from 4.274 to 43.706 g/kg body weight, which are higher than amsacrine (3.569 g/kg body weight). Regarding the rat maximum tolerated dose model, all candidates except 13a, 15a, 18, and 20 demonstrated maximum tolerated dose with values ranging from 0.070 to 0.241 g/kg body weight which are higher than that of amsacrine (0.068 g/kg body weight). For the rat oral LD 50 model, all compounds revealed oral LD 50 values lower than that of amsacrine (3.005 g/kg body weight). Additionally, all compounds were anticipated to be non-toxic against the developmental toxicity potential model. For rat chronic LOAEL model, compounds 11a, 12a, 13a, 14a, and 15a displayed LOAEL values ranging from 0.029 to 0.044 g/kg body weight. These values are higher than amsacrine (0.025 g/kg body weight).

Molecular dynamic simulation.
To analyze the trajectory obtained from the MD simulation, Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Solvent Accessible Surface Area (SASA), Radius of Gyration (RoG), and the change in the number of H-bonds were calculated using TCL scripts in VMD (Fig 12). Fig 12A showed the RMSD of the protein backbone along the trajectory. The protein showed a short stable fluctuation from 5 ns to 35 ns around 3.08 Å before rising to an average of 4.36 Å in the last 65 ns. This indicates that the system has stabilized. Fig 12B-12D give information about the folding and unfolding of the protein. The number of internal H-bonds, RoG, and SASA, show a stable trend during the whole simulation with an average of approximately 172 bonds, 32.8 Å, and 36335 Å 2 , respectively. Fig 12E showed the C α RMSF values which give an indication on the average fluctuation of each amino acid during the simulation. Some regions of the protein showed high fluctuations such as both the C and N terminals and loops within the protein as they are not included in a secondary structure.
Since this system includes DNA molecule and ligand, the center of mass (COM) distance between the ligand and the protein and the distance between the DNA and the protein were measured to see whether either of them moves away from the protein (Fig 13). The distance between the COM of the protein and DNA (red line) showed a stable trend along the trajectory with an average of 15.1 Å. On the other hand, the COM distance between the ligand and the protein showed stability starting from 25 ns with an average of 19.5 Å. The trajectory was clustered using TTClust library and to obtain a representative frame for each cluster. Two clusters were obtained from the trajectory and were used with PLIP webserver to get the interacting amino acids and the types of interactions ( Table 6). Only two types of interactions are present: H-bond (three interactions) and hydrophobic interactions (two interactions).   docking studies revealed that the docked compounds have a similar binding mode to that of doxorubicin with binding energies ranging from-29.41 to-46.03 kcal/mol. Finally, the synthesized candidates had good toxicity profiles and may serve as lead compounds for the discovery of new anticancer agents.
4.1.2. General procedure for the synthesis of compounds 11 a,b -20. Compounds 7a, b, and 10 (1.8 mmol) were dissolved in isopropyl alcohol (20 mL) and then added to appropriate amine (3.6 mmol). The mixture was refluxed at 120˚C for 6-10 h. After the end of the reaction (monitored by TLC), the mixture was allowed to cool. Water (200 mL) was added to the reaction mixture and the formed precipitate was collected by filtration and dried. The solid mass

Biological evaluation
4.2.1. In vitro anti-proliferative activity. The anti-proliferative activity of the synthesized compounds was assessed using MTT assay protocol [26][27][28]56]. A panel of human cancer cell lines namely, colorectal carcinoma (HCT-116) and hepatocellular carcinoma (HepG2) was used in this test. Doxorubicin was used as a positive control. The cell lines were got from ATCC (American Type Culture Collection) via the Holding company for biological products and vaccines (VACSERA, Cairo, Egypt). The anti-proliferative activities of the tested compounds were determined quantitatively as follows: At first, the cells were cultured into a medium of RPMI-1640 with 10% fetal bovine serum. Then, two different antibiotics were added at 37˚C in a 5% CO2 incubator: penicillin (100 units/mL) and streptomycin (100 μg/mL). Next, we seeded the cells in a 96-well plate by a density of 1.0 x 104 cells / well at 37˚C for 48 h under 5% CO2. The synthesized compounds with different concentrations were applied into the cell lines and incubated for 48 h. After 48 h, 20 μl of MTT solution (5mg/mL) was added and incubated for 4 h. Then, DMSO (100 μl) was added into each well to dissolve the formed purple formazan. After that, a colorimetric assay was measured and recorded at absorbance of 570 nm using a plate reader (EXL 800, USA). The relative cell viability in percentage was calculated as (A570 of treated samples/A570 of untreated sample) X 100. Results for IC50values of the active compounds were summarized in Table 1 14a, 16, 17, 18, 19, and 20) were analyzed for their Topo II inhibitory activities. The reported method described by Patra et al [57] was applied using Topo II drug screening kit (TopoGEN, Inc., Columbus). Doxorubicin was used as a positive control.
A typical enzyme reaction was structured to determine Topo II activity. The reaction mixture included Topo II (2 μl), substrate super coiled pHot1 DNA (0.25 μg), 50 μg/ml test compound (2 μl), and assay buffer (4 μl). To start the reaction, the mixture was allowed to incubate in 37˚C for 30 min. To terminate the reaction, a mixture of 10% sodium dodecylsulphate (2 μl) and proteinase K (50 μg/mL) was added at 37˚C for 15 min. then incubated for 15 min at 37˚C.
After that, the DNA was run on 1% agarose gel in BioRad gel electrophoresis system for 1-2 h followed by staining with GelRedTM stain for 2 h and destained for 15 min with TAE buffer. The gel was imaged via BioRad's Gel DocTMEZ system. Both supercoiled and linear strands DNA were incorporated in the gel as markers for DNA-Topo II intercalators. The results of IC 50 values were calculated using the GraphPad Prism version 7. Each reaction was performed in duplicate, and at least three independent determinations of each IC 50 were made.

DNA/Methyl green assay.
The most active anti-proliferative members (13a, 14a,  16, 17, 18, 19, and 20) were evaluated for their DNA-binding affinities, using doxorubicin as a positive control according to methyl green dye method described by Burres et al [29]. Activated Calf Thymus DNA (Merk, Germany) was treated with methyl green (Merk, Germany), then the synthesized compounds were applied to displace the methyl green dye, producing equivalent color. The results were reported as a 50% inhibition concentration values (IC 50 ) calculated by linear regression of data plotted on a semi-log scale and summarized in Table 1.

Toxicity studies.
The toxicity parameters of the synthesized compounds were calculated using Discovery studio 4.0. Sorafenib was used as a reference drug. At first, the CHARMM force field was applied then the compounds were prepared and minimized according to the preparation of small molecule protocol. Then different parameters were calculated from toxicity prediction (extensible) protocol [70]